The Search for Single-Domain Antibodies Interacting with the Receptor-Binding Domain of SARS-CoV-2 Surface Protein.

We performed a search for nanoantibodies that specifically interact with the receptor-binding domain (RBD) of the SARS-CoV-2 surface protein. The specificity of single-domain antibodies from the blood sera of a llama immunized with RBD of SARS-CoV-2 surface protein S (variant B.1.1.7 (Alpha)) was analyzed by ELISA. Recombinant trimers of the SARS-CoV-2 spike protein were used as antigens. In this work, a set of single-domain antibodies was obtained that specifically bind to the RBD of the SARS-CoV-2 virus.

[Impact of neutron radiation on the viability of tumor cells cultured in the presence of boron-10 isotope].

Objective: To investigate the impact of a neutron beam formed with the accelerator-based epithermal neutron source designed at the G.I. Budker Institute of Nuclear Physics (INP) on the viability of human and animal tumor cells cultured in the presence of boron-10 isotope. Material and Methods: Human U251 and T98G glioma cells and Chinese hamster CHO-K1 and V-79 cells were incubated at various concentrations in the culture medium containing 10B-enriched L-boronophenylalanine. The cells were irradiated with a neuron beam using the accelerator-based epithermal neuron source. A clonogenic assay was used to evaluate the viability of the irradiated cells. The absorbed doses obtained from elastic scattering of fast neutrons by substance nuclei and the doses obtained from boron neutron capture were calculated using the NMS code. The absorbed doses of gamma-radiation were measured with a mixed radiation dosimeter. Results: The viability of boron-containing and intact human U251 and T98G cell lines and Chinese hamster CHO-K1 and V-79 cells was analyzed after neutron beam radiation. Irradiation of all four cell lines were cultured in the presence of 10B was shown to reduce their colony-forming capacity compared with the control. Elevated boron levels in the culture medium resulted in a significant decrease in the proportion of survived cells. Radiation had the most pronounced impact on the proliferative capacity of the human U251 glioma cell lines. Conclusion: The cultures of human tumor cells and mammalian cells demonstrated that the neutron beam formed with the accelerator-based epithermal neutron source designed at the INP, was effective in reducing the viability of tumor cells in the presence of 10B.

[Expression of human B-cell specific receptor FCRL1 in normal individuals and in patients with autoimmune diseases].

The comparative analysis of expression level of FCRL1 gene encoding human B-cell surface receptor in healthy individuals and patients with autoimmune diseases was carried out. For the expression estimation we used results of DNA dot-hybridization on the membranes, containing cDNA samples from subpopulations of blood cells of patients with autoimmune diseases. The quantitative estimation of hybridization signals showed that expression level of FCRL1 gene in peripheral blood B-lymphocytes was significantly higher in patients with a multiple sclerosis, lupus anticoagulans, Takayasu's arteritis and also in von Willebrand disease than in healthy individuals. FCRL1-specific monoclonal and polyclonal antibodies were raised. They were proven to detect FCRL1 in Western blotting, immunohistochemistry and flow cytometry. It was found that FCRL1 is expressed on the surface of CD19+ mature B-cells. In tonsil FCRL1-positive cells were located in crypt area: in mantle zone of secondary lymphoid follicles and among cells of lymphoepithelium. FCRL1-positive cells were also found in B-cell follicles of the spleen.

[MHC-multimers and their application in studies of antiviral immune response].

Application of main histocompatibility complex tetrames (MHC-tetramers) for antigen specific T-cells detection and analysis coupled with flow cytometry opened new opportunities for T-cell response analysis. MHC-multimers allow the detection of T-cells against viral, cancer and vaccine antigens with exceptional sensitivity and specificity. This approach has become the "gold standard" for quantative analysis of T-cell immune response. Certain aspects of analysis using MHC-tetramer are examined, and importance of this approach in T-cell response efficacy evaluation in anti-HIV vaccine trials as well as in HIV positive patients are discussed.

[Expression patterns of the human and mouse IFGP family genes].

The IFGP family is a recently identified group of human and mouse genes structurally related to the genes of leukocyte Fc-receptors. In this study we compared expression patterns of six human and four mouse IFGP genes. With the exception of mouse IFGP2, the genes of the family were found to be predominantly expressed in haemopoietic cells. Expression of human IFGP1-IFGP5 and mouse IFGP3 was B cell-specific. Mouse IFGP1 transcripts were mainly found in B cells, but this gene may be either expressed by nonlymphoid cells. Expression of the human IFGP6 was detected in CD8+ T cells and NK cells. We further demonstrated that alternative splicing of human IFGP1 and IFGP6 mRNA may generate transcripts coding for the previously unknown isoforms. The novel IFGP1 isoform lacks transmembrane domain, whereas the IFGP6 isoform has altered cytoplasmic tail. The data obtained indicate that the receptors of family may contribute to the regulation of development and/or functions of three effector types of lymphocytes, namely B cells, CD8 T cells and NK cells.

Cloning and characterization of the human FCRL2 gene.

We report cloning and characterization of FCRL2, a novel human gene that belongs to the FcR family. The gene is closely linked and structurally similar to the recently identified FCRL/FREB/FcRX gene. The encoded protein is composed of three Ig-like domains and a C-terminal mucin-like domain containing a conserved alpha-helical motif with dileucine signals. Intraexonic splicing may generate two alternative transcripts, coding for isoforms with the third and fourth domains replaced by entirely different amino acid sequences. Like FCRL, the full-length isoform of FCRL2 is expressed intracellularly in transfected 293T cells. Expression analysis revealed FCRL2 mRNA only in placenta. The gene transcripts were not detected in lymphoid tissues or in the main leukocyte subsets isolated from peripheral blood. However, we found that FCRL2 is differentially expressed by transformed B cell lines. Of interest is also the finding that the gene expression may be up-regulated in the progression of melanocytic tumors.

CD3epsilon homologues in the chondrostean fish Acipenser ruthenus.

CD3epsilon is an essential component of the T-cell receptor (TCR) complex for antigen. We report here molecular cloning and characterization of cDNAs encoding the CD3epsilon homologues in sterlet (Acipenser ruthenus), a representative of primitive chondrostean fishes. Sequence analysis of the cDNA clones demonstrated unexpectedly high CD3epsilon gene heterogeneity in this species. While some cDNAs encoded proteins with the structure typical of mammalian CD3epsilon, others coded for proteins lacking the membrane-proximal half of the extracellular domain. Two cDNAs contained in-frame stop codons in the region encoding the cytoplasmic domain. Based on genomic blot analysis and RT-PCR typing of individual spleen RNAs, we suggest that sterlet may possess two highly polymorphic CD3epsilon loci, of which one can produce alternatively spliced transcripts. The structural elements shown to be functionally important in the mammalian CD3epsilon are strongly conserved in the sterlet CD3epsilon. The cytoplasmic region contains an immunoreceptor tyrosine-based activation motif (ITAM) with YEPI and YSGL tyrosine-containing sequences that are characteristic of only this TCR subunit. The pattern of sequence conservation indicates also that strong selection pressure was imposed on a motif VYYW at the C-end of the transmembrane domain and on a CD3epsilon-specific proline-rich motif RXPPVP juxtaposed to the N-terminus of the ITAM. Weak similarity of the sterlet CD3epsilon with the chicken and Xenopus CD3gamma/delta indicates that these two TCR subunits diverged before radiation of bony fishes and tetrapods. While the role of CD3epsilon heterogeneity in sterlet remains to be elucidated, the data obtained show that the basic mechanisms of TCR signaling have ancient evolutionary origin.

Cloning of a CXCR4 homolog in chondrostean fish and characterization of the CXCR4-specific structural features.

The chemokine receptor CXCR4 and its ligand SDF-1 are essential components of hematopoiesis, organogenesis and immunomodulation in mammalian species. We cloned a cDNA encoding CXCR4 homolog of sterlet (Acipenser ruthenus), a representative of chondrostean fishes. The deduced amino acid sequence of sterlet CXCR4 is almost equally distant from mammalian and teleost CXCR4 (66-68% identical residues). Percent identity with the other chemokine receptors varies in the 30-35% range. The CXCR4 sequences from the three phylogenetically diverged lineages were compared with the sequences of the other chemokine receptors to determine the CXCR4-specific structural elements that were conserved during vertebrate evolution. The characteristic residues and/or motifs are located predominantly in the intracellular and extracellular regions and in the third, fourth and fifth transmembrane domains. The data presented may be helpful for structure-function analysis of the CXCR4 ligand binding and signal transduction.

Identification of an IL-8 homolog in lamprey (Lampetra fluviatilis): early evolutionary divergence of chemokines.

Subtractive hybridization was used to study river lamprey (Lampetra fluviatilis) leukocyte-specific cDNA. A clone representing the most abundant component (12%) of the leukocyte library subtracted with liver cDNA was isolated and characterized. The cDNA encodes a presumably secreted polypeptide of 101 residues. The 3' untranslated region of the cDNA contains motifs characteristic of the transiently expressing genes. Comparison of the deduced amino acid sequence with known protein sequences revealed its homology to the members of the chemokine superfamily. Designated as LFCA-1, the lamprey protein contains four conserved cysteines, of which the first two are separated by a residue, and a number of other CXC family characteristic residues. LFCA-1 has the highest similarity to the chicken EMF-1 (40%) and to the mammalian IL-8 (32-33%). However, it lacks the ELR motif essential for the function of the mammalian IL-8-related chemokines. Based on the phylogenetic analysis of the LFCA-1 relationship to the higher vertebrate chemokines, it is concluded that the evolutionary origin of the chemokine superfamily is ancient, and that the divergence of the CXC and CC families most likely occurred at the time or before the first vertebrates emerged.

Expression of immunoglobulin kappa and lambda chains in mink.

The ratio of kappa and lambda chains of immunoglobulins varies significantly from one species to another. It has previously been thought that lambda was only type expressed in mink. We tested mink immunoglobulin light chains using two monoclonal antibodies G80 and G88. It has been shown that G80 and G88 specifically recognize two antigenically different subpopulations of the light chains. Immunochemical analysis of these subpopulations separated by affinity chromatography suggested that they represent lambda and kappa types of light chains, respectively. Screening of a mink cDNA library with monoclonal antibody G88 resulted in the isolation of clone pIGK-1 containing kappa chain-encoding sequence. The cDNA insert of pIGK-1 included most of the V segment, as well as the J, C and 3' untranslated sequences. Mink V kappa sequence shown the highest homology with the human V kappa II subgroup genes (76-79%). Mink C kappa sequence was 53-63% homologous to C kappa of other species. The striking feature of mink C kappa chain is the presence of glutamine in the C-terminal position. Southern blot analysis suggested that mink haploid genome has one C kappa gene and multiple V kappa genes. The kappa:lambda chain ratio in the 12 minks studied was, on the average, 46:54. The same ratio was observed for the kappa- and lambda-producing cells in the mesenteric lymph nodes. The five previously identified mink light chain allotypes were assigned to the lambda chains, thereby confirming that lambda chains in this species are additionally subdivided into several subtypes.

Monoclonal antibodies against heavy and light chains of domestic mink IgG.

A panel of 26 monoclonal antibodies (MAbs) specific to mink IgG was produced and analyzed by ELISA, immunodiffusion assay (IDA) and immunoblotting assay. All the raised MAbs were directed against the isotypic IgG epitopes. Immunoblotting assay demonstrated that 11 MAbs reacted only with the Fc-fragments of IgG and 7 only with the light chains. Four antibodies bound to the Fab-containing fragments and failed to react with the Fc-fragments or isolated L-chains. Three MAbs did not react with IgG in IDA. Based on the results of IDA and cross-blocking assays, the MAbs were divided into 10 groups, with the MAbs of each group recognizing the same epitope. In IDA some MAbs were able to react with the epitopes which are common to the IgGs of some other representatives of Mustelidae family and also to some mammalian species remote from mink (dog, horse, pig, fox and rabbit).

Genetic polymorphism of IgG in the mink. IX. High proportion of allotype-producing lymphocytes in individuals with minor level of allotypes H3 and H4 in serum.

The levels of mink C gamma-allotypes (H3, H4, H6 and H8) were determined in sera, and the proportion of the corresponding allotype-synthesizing B cells was estimated in peripheral blood, spleen and mesenteric lymph nodes. Individual differences in H6 levels and, possibly, those in H8 were entirely dependent on the proliferation degree of the corresponding clone of B cells and also determined by the dosage of the structural gene. There was no correspondence between the great numbers of H3+, H4+ cells and low levels of H3 and H4 allotypes in the sera of the majority of minks with their minor expression. A possible cause of this discrepancy may be a blockade of the secretion of IgG by H3+, H4+ cells. There exists most likely a gene (or genes) controlling the blockade of IgG secretion. The regulation of C gamma-allotype expression is presumably effected in a manner specific to each of the allotypes.

Genetic polymorphism of IgG in the mink. VIII. A quantitative study of the expression of C gamma-allotypes (H3, H4, H6, H8) in sera.

The results of a quantitative study of the expression of mink C gamma-allotypes (H3, H4, H6, and H8) in sera are presented. H6 and H8 were found to be stably expressed, and the individual concentrations of the allotypes varied within one order of magnitude. Gene dosage effects were observed for H6 and H8: average sera allotype concentrations in homozygotes were twice those in heterozygotes. In contrast, the serum concentrations of H3 and H4 varied by three orders of magnitude, ranging from minor (2-200 micrograms/ml) to high (1-10 mg/ml). No gene dosage effects were observed for the expression of H3 and H4. Histograms for the population of H3 concentrations showed three peaks, sharply differing from those of H4, H6, and H8. There was no association between the minor expression of H3 and H4. The data obtained indicate that the expression of mink C gamma-allotypes is regulated by different allotype-specific mechanisms.

Mink-mouse hybridomas that secrete mink immunoglobulin G.

Optimum conditions were established to obtain mink-mouse interspecific hybridomas secreting mink IgG in fusions of mouse myelomas with mink immune spleen cells. Minks were immunized with allogeneic IgG, and the spleen cells were fused with three mouse myeloma lines P3-X63-Ag8.653, NSO and Sp2/0-Ag14. Of these, P3-X63-Ag8.653 and NSO were found to be the best fusion partners giving the highest yield of hybrid clones and number of IgG secreting clones. Cloning of mink-mouse hybridomas was efficient when BALB/c nu/nu peritoneal and spleen cells were used as feeders. The ten clonal lines produced secreted intact mink IgG molecules as shown by SDS-PAGE and subsequent immunoblotting. The secretion level of IgG ranged from 5 to 200 ng/ml in the clonal lines.

Genetic polymorphism of IgG in the mink. III. Instability of expression and the problem of the genetic control of C gamma-allotypes.

Quantitative expression of C gamma-allotype H4 of mink immunoglobulins was studied by enzyme-linked immunosorbent assay. The results presented suggest that production of H4 is under specific regulation. the concentration of H4 varies three orders of magnitude (10-10,000 micrograms/ml) from one mink to another. Fifteen percent of the sera of normal minks have the low H4 concentration, undetectable by the standard procedure of double immunodiffusion routinely used to test mink IgG allotypes. However, expression of these 'minor' allotypes may be significantly enhanced by hyperimmunization. Instability of this kind seems to be the main cause of earlier described deviations from Mendelian inheritance of C gamma-allotypes H2, H3 and H4.